Abstract There is ample evidence that melanoma patients (MPs) develop immune responses directed against tumor- associated antigens. However, high frequencies of tumor antigen-specific cytotoxic T-lymphocytes (CTL) often fail to induce tumor rejection. Among the numerous mechanisms of tumor-induced immunosuppression that contribute to the resistance of tumors to CTL responses, it is now well established that inhibitory receptors like PD1 play a critical role in dampening T cell responses to tumors. As a result, therapy with blocking anti-PD1 monoclonal antibodies has become one of the most potent therapies of melanoma, providing prolonged clinical benefits to 30-40% advanced MPs. There is strong evidence that pre-existing CD8+ tumor-infiltrating T cells correlate with clinical antitumor response to PD1 blockade. However, not all T cell-inflamed tumors respond to PD1 blockade and not all melanomas are inflamed. Hence, the mechanisms supporting response or resistance to PD1 blockade remain to be precisely determined. Two studies have shown the role of the gut microbiome in regulating clinical responses to CTLA4 and PD1 blockade in murine melanoma model. In addition, two studies in NSCLCs and renal cancers (RC), and melanoma patients (MPs), respectively, have shown that the presence of certain commensals correlated with better clinical outcome upon PD1 blockade. Fecal microbiota transplant (FMT) from PD1 responders (PD1Rs) in melanoma-bearing GF mice reduced significantly tumor growth as compared to FMT from PD1 non-responders (PD1NRs). We have observed the increased abundance of certain bacterial commensals in stools of PD1Rs as compared to PD1NRs, albeit significantly different from the ones recently published. The striking differences between our findings and the recent published study are not totally surprising when one considers the greater complexity and inter-individual variability of the human gut microbiota upon many host-dependent variables. It is therefore critically important to expand these studies in a larger number of MPs using state-of-the-art omic approach (metagenomics) and Systems Biology to evaluate the direct causality between commensal bacteria and clinical outcome. Here, we propose to test whether the gut microbiome modulates immune and clinical responses to PD1 blockade in the context a novel clinical trial with FMT obtained from PD1Rs combined with PD1 blockade in PD1NRs. This project will test the novel hypotheses that: 1) PD1Rs exhibit distinct gut microbiota profiles in terms of diversity, abundance and dynamics over time compared with PD1NRs; 2) the administration of single PD1R-derived FMT to a PD1NRs together with PD1 blockade promotes clinical antitumor response; and 3) the occurrence of clinical responses in MPs treated with FMT and PD1 blockade correlates with T cell responses to melanoma. This proposal will take advantage of the well-established and cross-disciplinary expertise of the investigators in melanoma, human cancer immunology, gut microbiome and metaomics, biostatistics, and systems biology.